Methods and articles for strand-specific polynucleotide detection with conjugated polymers

ABSTRACT

The invention further relates to conjugated polymers, and methods, articles and compositions employing them as described herein. In some aspects, the invention relates to methods, articles and compositions for the detection and analysis of biomolecules in a sample. Provided assays include those determining the presence of a target biomolecule in a sample or its relative amount, or the assays may be quantitative or semi-quantitative. The methods can be performed on a substrate. The methods can be performed in an array format on a substrate, which can be a sensor. In some embodiments, detection assays are provided employing sensor biomolecules that do not comprise a fluorophore that can exchange energy with the cationic multichromophore. In some aspects biological assays are provided in which energy is transferred between one or more of the multichromophore, a label on the target biomolecule, a label on the sensor biomolecule, and/or a fluorescent dye specific for a polynucleotide, in all permutations. The multichromophore may interact at least in part electrostatically with the sensor and/or the target, and an increase in energy transfer with the polymer may occur upon binding of the sensor and the target. Other variations of the inventions are described further herein.

STATEMENT REGARDING CONTINUING APPLICATIONS

This application claims priority as a continuation of U.S. patentapplication Ser. No. 11/329,861, filed Jan. 10, 2006, now U.S. Pat. No.7,811,755, issued Oct. 12, 2010, which claims the benefit of U.S.Provisional Application No. 60/642,883 filed Jan. 10, 2005; whichapplications are hereby incorporated by this reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Nos. 0343312and GM62958-01 awarded by the National Science Foundation and theNational Institutes of Health. The U.S. Government may have limitedrights in this invention.

TECHNICAL FIELD

This invention relates to methods, articles and compositions for thedetection and analysis of biomolecules in a sample. The inventionfurther relates to conjugated polymers and methods, articles andcompositions employing them as described herein.

BACKGROUND OF THE INVENTION

Methods for the detection of biomolecules such as nucleic acids arehighly significant not only in identifying specific targets, but also inunderstanding their basic function. Hybridization probe technologies inparticular continue to be one of the most essential elements in thestudy of gene-related biomolecules.^(1,2,3) They are useful for avariety of both commercial and scientific applications, including theidentification of genetic mutations or single nucleotide polymorphisms(SNP's), medical diagnostics, gene delivery, assessment of geneexpression, and drug discovery.^(4,5,6,7) Heterogeneous formats forperforming such hybridization probe assays have become increasinglycommon and powerful with the advancement of gene chip and DNA microarraytechnologies.^(8,9,10,11) Such systems allow for high throughputscreening of hundreds to thousands of genes in a single experiment.

There is a continuing need in the art for methods of detecting andanalyzing particular biomolecules in a sample, and for compositions andarticles of manufacture useful in such methods. There is a need in theart for novel CCPs, for methods of making and using them, and forcompositions and articles of manufacture comprising such compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Use of cationic conjugated polymers (CCPs) for the detection ofPNA/DNA hybridization. The CCP is shown in orange, the PNA-C* in black,the non complementary ssDNA in green and the complementary ssDNA in red.

FIG. 2. Normalized absorption (a and c) and photoluminescence (b and d)spectra of PFBTpoly[9,9′-bis(6″-N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole)dibromide] in buffer (in black) and in films (in blue). Absorption (e)and emission (f) of PNA_(I)-Cy5 are shown in red. Photoluminescencespectra were obtained by excitation at 460 nm for PFBT and at 645 nm forCy5. The buffer is 25 mM phosphate buffer, pH=7.4.

FIG. 3. Photoluminescence spectra of PFBT with (a) hybridizedPNA_(I)-Cy5/ssDNA_(I)c and (b) non-hybridized PNA_(I)-Cy5+ssDNA_(I)n in25 mM phosphate buffer with 5% NMP (excitation wavelength=460 nm,[PNA_(I)-Cy5]=1.0×10⁻⁹ M). Residual polymer emission was subtracted forclarity.

FIG. 4 depicts an embodiment of a surface bound PNA probe structure.

FIG. 5. Amplification of a PNA (black)/ssDNA-C* (red) solid-state sensorby polyelectrolytic deposition of the CCP (orange).

FIG. 6. Fluorescence spectra of (a) ssDNA_(II)c-Cy5/PNA_(II)/PFBT(excitation at 470 nm), (b) ssDNA_(II)n-Cy5/PNA_(II)/PFBT (excitation at470 nm), (c) ssDNA_(II)c-Cy5/PNA_(II)/PFBT (direct excitation of Cy5 at645 nm).

FIG. 7. Hybridization of PNA (a) with ssDNA results in an increase ofnegative charge at the surface (b). Electrostatic interactions result inadsorption of the CCP (c).

FIG. 8. Comparison of PFBT intensity after 0.5 μL addition to (a)PNA_(II)/ssDNA_(II)c and (b) PNA_(II)/ssDNA_(II)n surfaces, followed bywashing. The initial PFBT concentration is 5×10⁻⁷ M.

FIG. 9. Block diagram showing a representative example logic device.

FIGS. 10A and 10B. Block diagrams showing representative examples of akit.

FIG. 11: (A) Emission profile of a DNA probe slide hybridized with Alexa750 labeled complimentary DNA. Panel A showed the profile of all thespots on the slide. The top two rows had only probe DNA but not theAlexa 750 labeled target and the bottom three rows had both the probeDNA and the hybridized Alexa 750 labeled target. Panel B showed theemission of Alexa 750 at 780 nm when excited at a wavelength of 633 nm.Panel C showed the polymer emission at 578 nm when excited at awavelength of 488 nm. Panel D showed the fluorescence energy transfer(FRET) of the polymer PFBT to Alexa 750. In Panel D the excitationwavelength used was 488 nm and emission wavelength used was 780 nm. (B)Comparison of Alex 750 and FRET signal intensity. Spots 1 to 6 (uppertwo rows in FIG. 11A) had no Alexa 750 labeled target. Spots 7 to 15(lower two rows in FIG. 11A) were hybridized Alexa 750 labeled target.The Alexa 750 signals generated by FRET were over 50% more intense thanthose generated by direct Alexa 750 excitation.

FIG. 12: MWG genome starter arrays tested with PFBT polymer usingAlexa647 labeled cDNA. (A) Array image obtained on a Perkin ElmerProScanArray. Alexa647 signals were obtained with an excitationwavelength of 633 nm and an emission wavelength of 670 nm using a lasersetting of 80% and a PMT gain setting of 50%. Polymer signals wereobtained with an excitation wavelength of 488 nm and an emissionwavelength of 578 nm using a laser setting of 80% and a PMT gain settingof 35%. FRET signals were obtained with an excitation wavelength of 488nm and an emission wavelength of 670 nm using a laser setting of 80% anda PMT gain setting of 35%. (B) Mean signal intensity of the Alexa647spots relative to the Alexa647 spots excited using energy transfer fromthe PFBT polymer (FRET). A crosstalk correction between the PFBT andAlexa647 emission was performed by subtracting ⅕ of the Polymer signalfrom the FRET signal. The FRET response is correlated with the amount ofbound target labeled with Alexa647.

DETAILED DESCRIPTION OF THE INVENTION

Homogeneous DNA assays with increased sensitivity, relative to smallmolecular counterparts, have recently appeared that take advantage ofthe optical amplification afforded by conjugated polymers.¹² Theemission intensities of acceptor chromophores on hybridization probesare magnified, relative to direct excitation, when the absorptioncoefficient of the polymer is large and the fluorescence resonanceenergy transfer (FRET) from the polymer to the acceptor is efficient.¹³Conjugated polymers offer other transduction mechanisms, for instancetheir optical properties can be modified upon complexation with doublestranded or single stranded DNA.¹⁴

One successful DNA sensing method involves the use of labeled peptidenucleic acids (PNAs) and cationic conjugated polymers (CCPs). The assay,shown in FIG. 1, takes advantage of electrostatic interactions typicalof oppositely charged polyelectrolytes.¹⁵ There are three components inthe assay: a CCP (shown in orange), a PNA-C* (shown in black), where C*is a reporter fluorophore, and the negatively charged target singlestranded DNA (ssDNA), which may be complementary (in red) ornon-complementary (in green) to the PNA sequence. The PNA-C* and thessDNA are first treated according to hybridization protocols and the CCPis added to the resulting solution. If the ssDNA does not hybridize, oneencounters situation A in FIG. 1, where the ssDNA and the CCP arebrought together by non specific electrostatic forces. The PNA-C* is notincorporated into the electrostatic complex. Situation B shows that whenthe PNA-C* and the complementary ssDNA hybridize, the CCP binds to theduplex structure. If the optical properties of the CCP and the C* areoptimized, then selective excitation of the CCP results in veryefficient FRET to C*. One can therefore monitor the presence of specificssDNA sequences by monitoring the C* emission or the CCP to C* emissionratio. Although FIG. 1 is specific to PNA/DNA interactions, othersimilar assays have appeared that incorporate peptide/RNA, DNA/DNA andRNA/RNA recognition pairs.¹⁶

A strand-specific polynucleotide sensing method is described based onsurface bound peptide nucleic acids (PNAs) and water-soluble cationicconjugated polymers (CCPs). The main transduction mechanism operates bytaking advantage of the net increase in negative charge at the PNAsurface, which occurs upon ssDNA hybridization. Electrostatic forcescause the oppositely charged CCP to bind selectively to the“complementary” surfaces. This approach circumvents the current need tolabel the probe or target strands. The polymer used in these assays ispoly[9,9′-bis(6″-N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole)dibromide] (PFBT), which was specifically designed and synthesized to becompatible with excitation sources used in commonly used DNA microarrayreaders. Furthermore, the utility of PFBT has been demonstrated inhomogenous and solid-state assays that involve fluorescence resonanceenergy transfer (FRET) to a reporter dye (Cy5) and that can benefit fromthe light harvesting properties observed in water soluble conjugatedpolymers.

The inventions described herein are useful for any assay in which asample can be interrogated regarding a target biomolecule. Typicalassays involve determining the presence of the target biomolecule in thesample or its relative amount, or the assays may be quantitative orsemi-quantitative.

The methods can be performed on a substrate. The assay can be performedin an array format on a substrate, which can be a sensor. Thesesubstrates may be surfaces of glass, silicon, paper, plastic, or thesurfaces of optoelectronic semiconductors (such as, but not confined to,indium-doped gallium nitride or polyanilines, etc.) employed asoptoelectronic transducers. The location of a given sensorpolynucleotide may be known or determinable in an array format, and thearray format may be microaddressable or nanoaddressable.

The methods of the invention can all be performed in multiplex formats.A plurality of different sensor polynucleotides can be used to detectcorresponding different target polynucleotides in a sample through theuse of different signaling chromophores conjugated to the respectivesensor polynucleotides or through the use of localization of particularsensor polynucleotides to determinable regions of the substrate.Multiplex methods are provided employing 2, 3, 4, 5, 10, 15, 20, 25, 50,100, 200, 400 or more different sensors which can be used simultaneouslyto assay for corresponding different target polynucleotides.

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to the particularmethodology, devices, solutions or apparatuses described, as suchmethods, devices, solutions or apparatuses can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention.

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “a target polynucleotide” includes a plurality of targetpolynucleotides, reference to “a sensor polynucleotide” includes aplurality of sensor polynucleotides, and the like. Additionally, use ofspecific plural references, such as “two,” “three,” etc., read on largernumbers of the same subject less the context clearly dictates otherwise.

Terms such as “connected,” “attached,” “conjugated” and “linked” areused interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise. Where a range of values is recited, it is tobe understood that each intervening integer value, and each fractionthereof, between the recited upper and lower limits of that range isalso specifically disclosed, along with each subrange between suchvalues. The upper and lower limits of any range can independently beincluded in or excluded from the range, and each range where either,neither or both limits are included is also encompassed within theinvention. Where a value being discussed has inherent limits, forexample where a component can be present at a concentration of from 0 to100%, or where the pH of an aqueous solution can range from 1 to 14,those inherent limits are specifically disclosed. Where a value isexplicitly recited, it is to be understood that values which are aboutthe same quantity or amount as the recited value are also within thescope of the invention, as are ranges based thereon. Where a combinationis disclosed, each subcombination of the elements of that combination isalso specifically disclosed and is within the scope of the invention.Conversely, where different elements or groups of elements aredisclosed, combinations thereof are also disclosed. Where any element ofan invention is disclosed as having a plurality of alternatives,examples of that invention in which each alternative is excluded singlyor in any combination with the other alternatives are also herebydisclosed; more than one element of an invention can have suchexclusions, and all combinations of elements having such exclusions arehereby disclosed.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods and materials are nowdescribed.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited. The publicationsdiscussed herein are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Alkyl” refers to a branched, unbranched or cyclic saturated hydrocarbongroup of 1 to 24 carbon atoms optionally substituted at one or morepositions, and includes polycyclic compounds. Examples of alkyl groupsinclude optionally substituted methyl, ethyl, n-propyl, isopropyl,n-butyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,n-heptyl, n-octyl, n-decyl, hexyloctyl, tetradecyl, hexadecyl, eicosyl,tetracosyl and the like, as well as cycloalkyl groups such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and norbornyl. The term “lower alkyl” refers toan alkyl group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.Exemplary substituents on substituted alkyl groups include hydroxyl,cyano, alkoxy, ═O, ═S, —NO₂, halogen, haloalkyl, heteroalkyl,carboxyalkyl, amine, amide, thioether and —SH.

“Alkoxy” refers to an “-Oalkyl” group, where alkyl is as defined above.A “lower alkoxy” group intends an alkoxy group containing one to six,more preferably one to four, carbon atoms.

“Alkenyl” refers to a branched, unbranched or cyclic hydrocarbon groupof 2 to 24 carbon atoms containing at least one carbon-carbon doublebond optionally substituted at one or more positions. Examples ofalkenyl groups include ethenyl, 1-propenyl, 2-propenyl (allyl),1-methylvinyl, cyclopropenyl, 1-butenyl, 2-butenyl, isobutenyl,1,4-butadienyl, cyclobutenyl, 1-methylbut-2-enyl, 2-methylbut-2-en-4-yl,prenyl, pent-1-enyl, pent-3-enyl, 1,1-dimethylallyl, cyclopentenyl,hex-2-enyl, 1-methyl-1-ethylallyl, cyclohexenyl, heptenyl,cycloheptenyl, octenyl, cyclooctenyl, decenyl, tetradecenyl,hexadecenyl, eicosenyl, tetracosenyl and the like. Preferred alkenylgroups herein contain 2 to 12 carbon atoms. The term “lower alkenyl”intends an alkenyl group of 2 to 6 carbon atoms, preferably 2 to 4carbon atoms. The term “cycloalkenyl” intends a cyclic alkenyl group of3 to 8, preferably 5 or 6, carbon atoms. Exemplary substituents onsubstituted alkenyl groups include hydroxyl, cyano, alkoxy, ═O, ═S,—NO₂, halogen, haloalkyl, heteroalkyl, amine, thioether and —SH.

“Alkenyloxy” refers to an “-Oalkenyl” group, wherein alkenyl is asdefined above.

“Alkylaryl” refers to an alkyl group that is covalently joined to anaryl group. Preferably, the alkyl is a lower alkyl. Exemplary alkylarylgroups include benzyl, phenethyl, phenopropyl, 1-benzylethyl,phenobutyl, 2-benzylpropyl and the like.

“Alkylaryloxy” refers to an “-Oalkylaryl” group, where alkylaryl is asdefined above.

“Alkynyl” refers to a branched or unbranched hydrocarbon group of 2 to24 carbon atoms containing at least one —C≡C— triple bond, optionallysubstituted at one or more positions. Examples of alkynyl groups includeethynyl, n-propynyl, isopropynyl, propargyl, but-2-ynyl,3-methylbut-1-ynyl, octynyl, decynyl and the like. Preferred alkynylgroups herein contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6, preferably 2 to 4, carbon atoms, andone —C≡C— triple bond. Exemplary substituents on substituted alkynylgroups include hydroxyl, cyano, alkoxy, ═O, ═S, —NO₂, halogen,haloalkyl, heteroalkyl, amine, thioether and —SH.

“Amide” refers to —C(O)NR′R″, where R′ and R″ are independently selectedfrom hydrogen, alkyl, aryl, and alkylaryl.

“Amine” refers to an —N(R′)R″ group, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Aryl” refers to an aromatic group that has at least one ring having aconjugated pi electron system and includes carbocyclic, heterocyclic,bridged and/or polycyclic aryl groups, and can be optionally substitutedat one or more positions. Typical aryl groups contain 1 to 5 aromaticrings, which may be fused and/or linked. Exemplary aryl groups includephenyl, furanyl, azolyl, thiofuranyl, pyridyl, pyrimidyl, pyrazinyl,triazinyl, biphenyl, indenyl, benzofuranyl, indolyl, naphthyl,quinolinyl, isoquinolinyl, quinazolinyl, pyridopyridinyl,pyrrolopyridinyl, purinyl, tetralinyl and the like. Exemplarysubstituents on optionally substituted aryl groups include alkyl,alkoxy, alkylcarboxy, alkenyl, alkenyloxy, alkenylcarboxy, aryl,aryloxy, alkylaryl, alkylaryloxy, fused saturated or unsaturatedoptionally substituted rings, halogen, haloalkyl, heteroalkyl, —S(O)R,sulfonyl, —SO₃R, —SR, —NO₂, —NRR′, —OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R,—(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′ where n is 0-4, and wherein R and R′are independently H, alkyl, aryl or alkylaryl.

“Aryloxy” refers to an “-Oaryl” group, where aryl is as defined above.

“Carbocyclic” refers to an optionally substituted compound containing atleast one ring and wherein all ring atoms are carbon, and can besaturated or unsaturated.

“Carbocyclic aryl” refers to an optionally substituted aryl groupwherein the ring atoms are carbon.

“Halo” or “halogen” refers to fluoro, chloro, bromo or iodo. “Halide”refers to the anionic form of the halogens.

“Haloalkyl” refers to an alkyl group substituted at one or morepositions with a halogen, and includes alkyl groups substituted withonly one type of halogen atom as well as alkyl groups substituted with amixture of different types of halogen atoms. Exemplary haloalkyl groupsinclude trihalomethyl groups, for example trifluoromethyl.

“Heteroalkyl” refers to an alkyl group wherein one or more carbon atomsand associated hydrogen atom(s) are replaced by an optionallysubstituted heteroatom, and includes alkyl groups substituted with onlyone type of heteroatom as well as alkyl groups substituted with amixture of different types of heteroatoms. Heteroatoms include oxygen,sulfur, and nitrogen. As used herein, nitrogen heteroatoms and sulfurheteroatoms include any oxidized form of nitrogen and sulfur, and anyform of nitrogen having four covalent bonds including protonated forms.An optionally substituted heteroatom refers to replacement of one ormore hydrogens attached to a nitrogen atom with alkyl, aryl, alkylarylor hydroxyl.

“Heterocyclic” refers to a compound containing at least one saturated orunsaturated ring having at least one heteroatom and optionallysubstituted at one or more positions. Typical heterocyclic groupscontain 1 to 5 rings, which may be fused and/or linked, where the ringseach contain five or six atoms. Examples of heterocyclic groups includepiperidinyl, morpholinyl and pyrrolidinyl. Exemplary substituents foroptionally substituted heterocyclic groups are as for alkyl and aryl atring carbons and as for heteroalkyl at heteroatoms.

“Heterocyclic aryl” refers to an aryl group having at least 1 heteroatomin at least one aromatic ring. Exemplary heterocyclic aryl groupsinclude furanyl, thienyl, pyridyl, pyridazinyl, pyrrolyl, N-loweralkyl-pyrrolo, pyrimidyl, pyrazinyl, triazinyl, tetrazinyl, triazolyl,tetrazolyl, imidazolyl, bipyridyl, tripyridyl, tetrapyridyl, phenazinyl,phenanthrolinyl, purinyl and the like.

“Hydrocarbyl” refers to hydrocarbyl substituents containing 1 to about20 carbon atoms, including branched, unbranched and cyclic species aswell as saturated and unsaturated species, for example alkyl groups,alkylidenyl groups, alkenyl groups, alkylaryl groups, aryl groups, andthe like. The term “lower hydrocarbyl” intends a hydrocarbyl group ofone to six carbon atoms, preferably one to four carbon atoms.

A “substituent” refers to a group that replaces one or more hydrogensattached to a carbon or nitrogen. Exemplary substituents include alkyl,alkylidenyl, alkylcarboxy, alkoxy, alkenyl, alkenylcarboxy, alkenyloxy,aryl, aryloxy, alkylaryl, alkylaryloxy, —OH, amide, carboxamide,carboxy, sulfonyl, ═O, ═S, —NO₂, halogen, haloalkyl, fused saturated orunsaturated optionally substituted rings, —S(O)R, —SO₃R, —SR, —NRR′,—OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R, —(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′where n is 0-4, and wherein R and R′ are independently H, alkyl, aryl oralkylaryl. Substituents also include replacement of a carbon atom andone or more associated hydrogen atoms with an optionally substitutedheteroatom.

“Sulfonyl” refers to —S(O)₂R, where R is alkyl, aryl, —C(CN)═C-aryl,—CH₂CN, alkylaryl, or amine.

“Thioamide” refers to —C(S)NR′R″, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Thioether” refers to —SR, where R is alkyl, aryl, or alkylaryl.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. These terms refer only to the primary structure of themolecule. Thus, the terms includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide.

Whether modified or unmodified, in some embodiments the targetnucleotide must have a polyanionic backbone, preferably asugar-phosphate background, of sufficient negative charge toelectrostatically interact with the polycationic multichromophore in themethods described herein, although other forces may additionallyparticipate in the interaction. The sensor polynucleotide is exemplifiedas a peptide nucleic acid, although other polynucleotides whichminimally interact with the multichromophore in the absence of targetcan be used. Suitable hybridization conditions for a given assay formatcan be determined by one of skill in the art; nonlimiting parameterswhich may be adjusted include concentrations of assay components, pH,salts used and their concentration, ionic strength, temperature, etc.

More particularly, the terms “polynucleotide,” “oligonucleotide,”“nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA,and mRNA, whether spliced or unspliced, any other type of polynucleotidewhich is an N- or C-glycoside of a purine or pyrimidine base, and otherpolymers containing a phosphate or other polyanionic backbone, and othersynthetic sequence-specific nucleic acid polymers providing that thepolymers contain nucleobases in a configuration which allows for basepairing and base stacking, such as is found in DNA and RNA. There is nointended distinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms are used interchangeably herein. These terms refer only to theprimary structure of the molecule. Thus, these terms include, forexample, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, andhybrids thereof including for example hybrids between DNA and RNA, andalso include known types of modifications, for example, labels,alkylation, “caps,” substitution of one or more of the nucleotides withan analog, internucleotide modifications such as, for example, thosewith negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (including enzymes (e.g. nucleases), toxins,antibodies, signal peptides, poly-L-lysine, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelates (of, e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, those with modified linkages (e.g.,alpha anomeric nucleic acids, etc.), as well as unmodified forms of thepolynucleotide or oligonucleotide.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides can also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or are functionalized asethers, amines, or the like. The term “nucleotidic unit” is intended toencompass nucleosides and nucleotides.

Furthermore, modifications to nucleotidic units include rearranging,appending, substituting for or otherwise altering functional groups onthe purine or pyrimidine base which form hydrogen bonds to a respectivecomplementary pyrimidine or purine. The resultant modified nucleotidicunit optionally may form a base pair with other such modifiednucleotidic units but not with A, T, C, G or U. Abasic sites may beincorporated which do not prevent the function of the polynucleotide;preferably the polynucleotide does not comprise abasic sites. Some orall of the residues in the polynucleotide can optionally be modified inone or more ways.

Standard A-T and G-C base pairs form under conditions which allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3and C4-NH2, of cytidine and the C2-NH2, N′—H and C6-oxy, respectively,of guanosine. Thus, for example, guanosine(2-amino-6-oxy-9-β-D-ribofuranosyl-purine) may be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine)results in a modified nucleotide which will not effectively base pairwith guanosine but will form a base pair with isoguanosine. Isocytosineis available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine maybe prepared by the method described by Switzer et al. (1993)Biochemistry 32:10489-10496 and references cited therein;2′-deoxy-5-methyl-isocytidine may be prepared by the method of Tor etal. (1993) J. Am. Chem. Soc. 115:4461-4467 and references cited therein;and isoguanine nucleotides may be prepared using the method described bySwitzer et al. (1993), supra, and Mantsch et al. (1993) Biochem.14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 toCollins et al. Other nonnatural base pairs may be synthesized by themethod described in Piccirilli et al. (1990) Nature 343:33-37 for thesynthesis of 2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione). Other suchmodified nucleotidic units which form unique base pairs are known, suchas those described in Leach et al. (1992) J. Am. Chem. Soc.114:3675-3683 and Switzer et al., supra.

“Complementary” or “substantially complementary” refers to the abilityto hybridize or base pair between nucleotides or nucleic acids, such as,for instance, between a sensor polynucleotide and a targetpolynucleotide. Complementary nucleotides are, generally, A and T (or Aand U), or C and G. Two single-stranded polynucleotides or PNAs are saidto be substantially complementary when the bases of one strand,optimally aligned and compared and with appropriate insertions ordeletions, pair with at least about 80% of the bases of the otherstrand, usually at least about 90% to 95%, and more preferably fromabout 98 to 100%.

Alternatively, substantial complementarity exists when a polynucleotideor PNA will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25bases, preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984).

“Preferential binding” or “preferential hybridization” refers to theincreased propensity of one polynucleotide or PNA to bind to itscomplement in a sample as compared to a noncomplementary polymer in thesample.

Hybridization conditions will typically include salt concentrations ofless than about 1M, more usually less than about 500 mM and preferablyless than about 200 mM. In the case of hybridization between a peptidenucleic acid and a polynucleotide, the hybridization can be done insolutions containing little or no salt. Hybridization temperatures canbe as low as 5° C., but are typically greater than 22° C., moretypically greater than about 30° C., and preferably in excess of about37° C. Longer fragments may require higher hybridization temperaturesfor specific hybridization. Other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, and the combination of parameters used is more importantthan the absolute measure of any one alone. Other hybridizationconditions which may be controlled include buffer type andconcentration, solution pH, presence and concentration of blockingreagents to decrease background binding such as repeat sequences orblocking protein solutions, detergent type(s) and concentrations,molecules such as polymers which increase the relative concentration ofthe polynucleotides, metal ion(s) and their concentration(s),chelator(s) and their concentrations, and other conditions known in theart.

“Multiplexing” herein refers to an assay or other analytical method inwhich multiple analytes can be assayed simultaneously.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not.

The Sample

The portion of the sample comprising or suspected of comprising thetarget biomolecule can be any source of biological material whichcomprises biomolecule that can be obtained from a living organismdirectly or indirectly, including cells, tissue or fluid, and thedeposits left by that organism, including viruses, mycoplasma, andfossils. In some embodiments, the target biomolecule comprises apolynucleotide. The sample may comprise a target polynucleotide preparedthrough synthetic means, in whole or in part. Typically, the sample isobtained as or dispersed in a predominantly aqueous medium. Nonlimitingexamples of the sample include blood, urine, semen, milk, sputum, mucus,a buccal swab, a vaginal swab, a rectal swab, an aspirate, a needlebiopsy, a section of tissue obtained for example by surgery or autopsy,plasma, serum, spinal fluid, lymph fluid, the external secretions of theskin, respiratory, intestinal, and genitourinary tracts, tears, saliva,tumors, organs, samples of in vitro cell culture constituents (includingbut not limited to conditioned medium resulting from the growth of cellsin cell culture medium, putatively virally infected cells, recombinantcells, and cell components), and a recombinant library comprisingpolynucleotide sequences.

The sample can be a positive control sample which is known to containthe target polynucleotide or a surrogate therefor. A negative controlsample can also be used which, although not expected to contain thetarget polynucleotide, is suspected of containing it (via contaminationof one or more of the reagents) or another component capable ofproducing a false positive, and is tested in order to confirm the lackof contamination by the target polynucleotide of the reagents used in agiven assay, as well as to determine whether a given set of assayconditions produces false positives (a positive signal even in theabsence of target polynucleotide in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify any target polynucleotide present orto render it accessible to reagents which are used in an amplificationscheme or to detection reagents. Where the sample contains cells, thecells can be lysed or permeabilized to release the polynucleotideswithin the cells. One step permeabilization buffers can be used to lysecells which allow further steps to be performed directly after lysis,for example a polymerase chain reaction.

The Target Biomolecule

A target biomolecule (e.g., a polysaccharide, a polynucleotide, apeptide, a protein, etc.) is employed that can bind to a sensorbiomolecule. The target may also interact at least in partelectrostatically with a polycationic multichromophore, which may be aconjugated polymer. In some embodiments, a target polynucleotide isemployed, and may be complementary to a sensor polynucleotide.

In embodiments where the target biomolecule is a polynucleotide, thetarget polynucleotide can be single-stranded, double-stranded, or higherorder, and can be linear or circular. Exemplary single-stranded targetpolynucleotides include mRNA, rRNA, tRNA, hnRNA, ssRNA or ssDNA viralgenomes, although these polynucleotides may contain internallycomplementary sequences and significant secondary structure. Exemplarydouble-stranded target polynucleotides include genomic DNA,mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viral genomes,plasmids, phage, and viroids. The target polynucleotide can be preparedsynthetically or purified from a biological source. The targetpolynucleotide may be purified to remove or diminish one or moreundesired components of the sample or to concentrate the targetpolynucleotide. Conversely, where the target polynucleotide is tooconcentrated for the particular assay, the target polynucleotide may bediluted.

Following sample collection and optional nucleic acid extraction, thenucleic acid portion of the sample comprising the target polynucleotidecan be subjected to one or more preparative reactions. These preparativereactions can include in vitro transcription (IVT), labeling,fragmentation, amplification and other reactions. mRNA can first betreated with reverse transcriptase and a primer to create cDNA prior todetection and/or amplification; this can be done in vitro with purifiedmRNA or in situ, e.g. in cells or tissues affixed to a slide. Nucleicacid amplification increases the copy number of sequences of interestsuch as the target polynucleotide. A variety of amplification methodsare suitable for use, including the polymerase chain reaction method(PCR), the ligase chain reaction (LCR), self sustained sequencereplication (3SR), nucleic acid sequence-based amplification (NASBA),the use of Q Beta replicase, reverse transcription, nick translation,and the like.

Where the target polynucleotide is single-stranded, the first cycle ofamplification forms a primer extension product complementary to thetarget polynucleotide. If the target polynucleotide is single-strandedRNA, a polymerase with reverse transcriptase activity is used in thefirst amplification to reverse transcribe the RNA to DNA, and additionalamplification cycles can be performed to copy the primer extensionproducts. The primers for a PCR must, of course, be designed tohybridize to regions in their corresponding template that will producean amplifiable segment; thus, each primer must hybridize so that its 3′nucleotide is paired to a nucleotide in its complementary templatestrand that is located 3′ from the 3′ nucleotide of the primer used toreplicate that complementary template strand in the PCR.

The target polynucleotide can be amplified by contacting one or morestrands of the target polynucleotide with a primer and a polymerasehaving suitable activity to extend the primer and copy the targetpolynucleotide to produce a full-length complementary polynucleotide ora smaller portion thereof. Any enzyme having a polymerase activity thatcan copy the target polynucleotide can be used, including DNApolymerases, RNA polymerases, reverse transcriptases, enzymes havingmore than one type of polymerase activity, and the enzyme can bethermolabile or thermostable. Mixtures of enzymes can also be used.Exemplary enzymes include: DNA polymerases such as DNA Polymerase I(“Pol I”), the Klenow fragment of Pol I, T4, T7, Sequenase® T7,Sequenase® Version 2.0 T7, Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli andPyrococcus sp GB-D DNA polymerases; RNA polymerases such as E. coli,SP6, T3 and T7 RNA polymerases; and reverse transcriptases such as AMV,M-MuLV, MMLV, RNAse H⁻ MMLV (SuperScript®), SuperScript® II,ThermoScript®, HIV-1, and RAV2 reverse transcriptases. All of theseenzymes are commercially available. Exemplary polymerases with multiplespecificities include RAV2 and Tli (exo-) polymerases. Exemplarythermostable polymerases include Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tliand Pyrococcus sp. GB-D DNA polymerases.

Suitable reaction conditions are chosen to permit amplification of thetarget polynucleotide, including pH, buffer, ionic strength, presenceand concentration of one or more salts, presence and concentration ofreactants and cofactors such as nucleotides and magnesium and/or othermetal ions (e.g., manganese), optional cosolvents, temperature, thermalcycling profile for amplification schemes comprising a polymerase chainreaction, and may depend in part on the polymerase being used as well asthe nature of the sample. Cosolvents include formamide (typically atfrom about 2 to about 10%), glycerol (typically at from about 5 to about10%), and DMSO (typically at from about 0.9 to about 10%). Techniquesmay be used in the amplification scheme in order to minimize theproduction of false positives or artifacts produced duringamplification. These include “touchdown” PCR, hot-start techniques, useof nested primers, or designing PCR primers so that they form stem-loopstructures in the event of primer-dimer formation and thus are notamplified. Techniques to accelerate PCR can be used, for examplecentrifugal PCR, which allows for greater convection within the sample,and comprising infrared heating steps for rapid heating and cooling ofthe sample. One or more cycles of amplification can be performed. Anexcess of one primer can be used to produce an excess of one primerextension product during PCR; preferably, the primer extension productproduced in excess is the amplification product to be detected. Aplurality of different primers may be used to amplify different targetpolynucleotides or different regions of a particular targetpolynucleotide within the sample.

Amplified target polynucleotides may be subjected to post amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe target polynucleotide prior to hybridization in order to providesegments which are more readily accessible. Fragmentation of the nucleicacids can be carried out by any method producing fragments of a sizeuseful in the assay being performed; suitable physical, chemical andenzymatic methods are known in the art.

An amplification reaction can be performed under conditions which allowthe sensor polynucleotide to hybridize to the amplification productduring at least part of an amplification cycle. When the assay isperformed in this manner, real-time detection of this hybridizationevent can take place by monitoring for light emission from thepolycationic multichromophore in the vicinity of the sensor duringamplification.

The Polycationic Multichromophore

Light harvesting multichromophore systems can efficiently transferenergy to nearby luminescent species. Mechanisms for energy transferinclude, for example, resonant energy transfer (Förster (orfluorescence) resonance energy transfer, FRET), quantum charge exchange(Dexter energy transfer) and the like. Typically, however, these energytransfer mechanisms are relatively short range, and close proximity ofthe light harvesting multichromophore system to the signalingchromophore is required for efficient energy transfer. Amplification ofthe emission can occur when the number of individual chromophores in thelight harvesting multichromophore system is large; emission from afluorophore can be more intense when the incident light (the “pumplight”) is at a wavelength which is absorbed by the light harvestingmultichromophore system and transferred to the fluorophore than when thefluorophore is directly excited by the pump light.

The multichromophores used in the present invention are polycationic sothat they can interact with a biomolecule comprising multiple anionicgroups, e.g. polysaccharides, polynucleotides, peptides, proteins, etc.In some embodiments, the multichromophore can interact with a targetpolynucleotide electrostatically and thereby bring a signalingchromophore on an uncharged sensor polynucleotide into energy-receivingproximity by virtue of hybridization between the sensor polynucleotideand the target polynucleotide. Any polycationic multichromophore thatcan absorb light and preferably emit or transfer energy can be used inthe methods described. Exemplary multichromophores that can be usedinclude conjugated polymers, saturated polymers or dendrimersincorporating multiple chromophores in any viable manner, andsemiconductor nanocrystals (SCNCs). The conjugated polymers, saturatedpolymers and dendrimers can be prepared to incorporate multiple cationicspecies or can be derivatized to render them polycationic aftersynthesis; semiconductor nanocrystals can be rendered polycationic byaddition of cationic species to their surface. In some embodiments, thepolycationic multichromophore is not detected by its ability to transferenergy when excited, and thus methods involving such detection schemesdo not require the multichromophore to emit or transfer energy.

In some embodiments, the multichromophore is a conjugated polymer (CP).More preferably, the CP is one that comprises “low bandgap repeat units”of a type and in an amount that contribute an absorption to the polymerin the range of about 450 nm to about 1000 nm. The low bandgap repeatunits may or may not exhibit such an absorption prior to polymerization,but does introduce that absorption when incorporated into the conjugatedpolymer. Such absorption characteristics allow the polymer to be excitedat wavelengths that produce less background fluorescence in a variety ofsettings, including in analyzing biological samples and imaging and/ordetecting molecules. Shifting the absorbance of the CP to a lower energyand longer wavelength thus allows for more sensitive and robust methods.Additionally, many commercially available instruments incorporateimaging components that operate at such wavelengths at least in part toavoid such issues. For example, thermal cyclers that perform real-timedetection during amplification reactions and microarray readers areavailable which operate in this region. Providing polymers that absorbin this region allows for the adaptation of detection methods to suchformats, and also allows entirely new methods to be performed.

Incorporation of repeat units that decrease the band gap can produceconjugated polymers with such characteristics. Exemplary optionallysubstituted species which result in polymers that absorb light at suchwavelengths include 2,1,3-benzothiadiazole, benzoselenadiazole,benzotellurodiazole, naphthoselenadiazole,4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxalines,low bandgap commercial dyes, olefins, and cyano-substituted olefins andisomers thereof.

For example, 2,7-carbazolene-vinylene conjugated polymers have beendescribed with peak absorptions ranging from about 455-485 nm (Morin etal., Chem. Mater. 2004, vol. 16, No. 23, pages 4619-4626). Polymers canbe prepared incorporating benzoselenadiazole with absorption maxima at485 nm. Similarly, polymers incorporating naphthoselenadiazole are knownwith absorption maxima at 550 nm. Polymers incorporating4,7-di(thien-2-yl)-2,1,3-benzothiadiazole are known with absorptionmaxima at about 515 nm. Polymers incorporating cyanovinylenes are knownwith peak absorptions in this region, for example from 372-537 nm, andexhibiting absorption above 700 nm (PFR(1-4)-S, Macromolecules, Vol. 37,No. 14, pages 5265-5273). Preparation of polymers incorporating monomersthat provide absorption in the spectral region up to 1000 nm has beendescribed (Ajayaghosh, A., et al., Chem. Soc. Rev., 2003, 32, 181; A.Ajayagosh and J. Eldo, Organic Letters, 2001, 3, 2595-2598.) Polymerssoluble in polar media and having absorptions from about 450 nm to about1000 nm can thus be synthesized by incorporation of low bandgap repeatunits and pendant polar or charged groups as described herein.

In some embodiments, the polymer is one whose absorbance is not shiftedsignificantly in the presence of target or anionic polynucleotide.Desirably, the polymer exhibits no more than about a 15 nm shift in peakabsorbance in the presence of target or anionic polynucleotide; thiscorresponds to no more than about a 0.08 eV shift for a peak absorptionof 480 nm. The polymer may exhibit a peak absorbance shift of about 20,15, 12, 10, 8 or 5 nm or less. The polymer may exhibit a peak absorbanceshift of about 0.10, 0.08, 0.06, 0.04 eV or less. This stability inabsorbance can provide desirable properties in bioassays. Polymers whoseabsorbance shifts excessively depending on assay conditions can lead toundesirable variability.

In some embodiments, a sufficient amount of an appropriate low bandgaprepeat unit is incorporated into the CP to render it capable ofabsorbing energy at a desired wavelength above 450 nm and providing adetectable signal, particularly when localized to a substrate by thesensor. The polymer may include 15 mol %, 20 mol %, 25 mol %, 30 mol %,35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, 95 mol %, or more of thelow bandgap repeat unit. In the case of benzothiadiazole-containingpolymers, typically greater than about 15 mol % of the repeat units arebenzothiadiazole. The exact number of such monomers in the final polymerwill depend on its overall length; efficient absorption dictates thatthe polymer have a plurality of the low bandgap repeat unit.

In some embodiments the polymer can amplify the signal from afluorophore to which it can transfer energy upon excitation. Forexample, an alternating copolymer of substituted fluorene monomers and2,1,3-benzothiadiazole (BT) monomers can provide a five-foldamplification in signal from a Cy-5 labeled sensor when excited at 460nm as compared to excitation of the fluorophore directly (see examples).As the light gathering abilities of the polymer at this wavelength aredirectly proportional to the amount of BT monomers, a polymer of thislength containing 10 mol % BT monomer would provide the same signal asthe fluorophore would in the absence of polymer, and there would be noamplification. Desirably, the polymer is of a length and comprises asufficient amount of low bandgap repeat units so that upon excitation ittransmits sufficient energy to a fluorophore so as to achieve a 50% orgreater increase in light emission from the fluorophore than can beachieved by direct excitation of the fluorophore in the absence ofpolymer.

In the case of copolymers of substituted fluorene and BT monomers, thiscan be accomplished by incorporating 15 mol % or more of BT monomers insuch a polymer. The exact amount of low bandgap repeat unit needed toprovide the desired degree of amplification is dependent on a number offactors, and may be determined empirically for a given monomer. Factorsto be considered include the length of the polymer, the molarabsorptivity of the monomer, and the interaction between the polymer andthe biomolecule with which it interacts. The polymer can desirably be ofa length and comprise a sufficient amount of an low bandgap repeat unitto provide a two-fold, three-fold, four-fold, five-fold, or greaterincrease in emission from a fluorophore to which it can transfer energy.

The CP can be a copolymer, and may be a block copolymer, a graftcopolymer, or both. A given repeat unit may be incorporated into thepolymer randomly, alternately, periodically and/or in blocks.

The particular size of the polymer is not critical, so long as it isable to absorb light in the relevant region. In some embodiments, thepolymer (which includes oligomers) also desirably is able to transferenergy to a fluorophore. In some embodiments the polymer has a molecularmass of at least about 1,000 Daltons to allow for efficient interactionwith a biomolecule. Typically the polymer will have a molecular mass ofabout 250,000 Daltons or less. An oligomer has at least two repeats of achromophoric unit, and may have a plurality of repeats including 3, 4, 5or more repeats. An oligomer can also comprise a combination of one ormore different chromophoric units, each of which independently may ormay not be repeated.

In some embodiments the polymer can have a high quantum yield. Thepolymer may have a quantum yield of greater than about 4% in solution,and may have a quantum yield of up to about 12%. The polymer may exhibita quantum yield in the solid state of about 4%.

In some embodiments, the polymer may comprise optionally substitutedfluorenyl monomers. Polymers comprising fluorenyl monomers exhibitingdesirable characteristics and are well studied. However, the absorptionprofile of fluorene monomers shows absorption at shorter wavelengthsthan is desired in some embodiments described herein. Thus fluorenecopolymers additionally incorporating monomers with lower bandgaps thanfluorene may be desirable in some applications described herein.

In some embodiments, the polymer may comprise optionally substitutedbiphenylene monomers. In some embodiments, the polymer may compriseoptionally substituted 2,7-carbazolenevinylene monomers.

The terms “monomer,” “monomeric unit” and “repeat unit” are used hereinto denote conjugated subunits of a polymer or oligomer. It is to beunderstood that the repeat units can be incorporated into the polymer atany available position(s) and can be substituted with one or moredifferent groups. Exemplary substituents on the repeat units can beselected from alkyl groups, C1-20 alkyl groups optionally substituted atone or more positions with S, N, O, P or Si atoms, C4-16 alkylcarbonyloxy, C4-16 aryl(trialkylsiloxy), alkoxy, C1-20 alkoxy, cyano,alkylcarbonyloxy, C1-20 alkylcarbonyloxy, and C1-20 thioether.

The CP contains a sufficient density of solubilizing functionalities torender the overall polymer soluble in a polar medium. Exemplary polarmedia include dimethylsulfoxide (DMSO), dimethylformamide (DMF),ethanol, methanol, isopropanol, dioxane, acetone, acetonitrile,1-methyl-2-pyrrolidinone, formaldehyde, water, and mixtures comprisingthese solvents. The CP is desirably soluble in at least one of thesepolar media, and may be soluble in more than one polar media. The CPpreferably contains at least about 0.01 mol % of the solubilizingfunctionality, and may contain at least about 0.02 mol %, at least about0.05 mol %, at least about 0.1 mol %, at least about 0.2 mol %, at leastabout 0.5 mol %, at least about 1 mol %, at least about 2 mol %, atleast about 5 mol %, at least about 10 mol %, at least about 20 mol %,or at least about 30 mol %. The CP may contain up to 100 mol % of thesolubilizing functionality, and may contain about 99 mol % or less,about 90 mol % or less, about 80 mol % or less, about 70 mol % or less,about 60 mol % or less, about 50 mol % or less, or about 40 mol % orless. Where monomers are polysubstituted, the CP may contain 200 mol %,300 mol %, 400 mol % or more solubilizing functionalities.

The polymers are desirably polycationic, and any or all of the subunitsof the polymer may comprise one or more cationic groups. Any suitablecationic groups may be incorporated. Exemplary cationic groups includeammonium groups, guanidinium groups, histidines, polyamines, pyridiniumgroups, and sulfonium groups.

Desirably, the CPs described herein are soluble in aqueous solutions andother polar solvents, and preferably are soluble in water. By“water-soluble” is meant that the material exhibits solubility in apredominantly aqueous solution, which, although comprising more than 50%by volume of water, does not exclude other substances from thatsolution, including without limitation buffers, blocking agents,cosolvents, salts, metal ions and detergents.

One synthetic approach to introducing a charged group into a conjugatedpolymer is as follows. A neutral polymer can be formed by the Suzukicoupling of one or more bis- (or tris- etc.) boronic acid-substitutedmonomers with one or more monomers that have at least two brominesubstitutions on aromatic ring positions. Bromine groups can also beattached to any or all of the monomers via linkers. Polymer ends can becapped by incorporation of a monobrominated aryl group, for examplebromobenzene. Conversion of the polymer to a cationic water-soluble formis accomplished by addition of condensed trimethylamine.

In some embodiments, the CCPs comprise one or more angled linkers with asubstitution pattern (or regiochemistry) capable of perturbing thepolymers' ability to form rigid-rod structures, allowing the CCPs tohave a greater range of three-dimensional structures. The angledlinker(s) are optionally substituted aromatic molecules having at leasttwo separate bonds to other polymer components (e.g., monomers, blockpolymers, end groups) that are capable of forming angles relative to oneanother which disrupt the overall ability of the polymer to form anextended rigid-rod structure (although significant regions exhibitingsuch structure may remain). The angled linkers may be bivalent orpolyvalent.

The angle which the angled linkers are capable of imparting to thepolymeric structure is determined as follows. Where the two bonds toother polymeric components are coplanar, the angle can be determined byextending lines representing those bonds to the point at which theyintersect, and then measuring the angle between them. Where the twobonds to other polymeric components are not coplanar, the angle can bedetermined as follows: a first line is drawn between the two ring atomsto which the bonds attach; two bond lines are drawn, one extending fromeach ring atom in the direction of its respective bond to the otherpolymeric component to which it is joined; the angle between each bondline and the first line is fixed; and the two ring atoms are then mergedinto a single point by shrinking the first line to a zero length; theangle then resulting between the two bond lines is the angle the angledlinker imparts to the CCP.

The angle which an angled linker is capable of imparting to the polymeris typically less than about 155°, and may be less than about 150°, lessthan about 145°, less than about 140°, less than about 135°, less thanabout 130°, less than about 125°, less than about 120°, less than about115°, less than about 110°, less than about 105°, less than about 100°,less than about 95°, less than about 90°, less than about 85°, less thanabout 80°, less than about 75°, less than about 70°, less than about65°, less than about 60°, less than about 55°, or less than about 50°.The angled linker may form an angle to its adjacent polymeric units ofabout 25°, 30°, 35°, 40°, 45°, 50°, 60° or more. The angled linker mayintroduce a torsional twist in the conjugated polymer, thereby furtherdisrupting the ability of the polymer to form a rigid-rod structure. Forangled linkers having an internally rotatable bond, such aspolysubstituted biphenyl, the angled linker must be capable of impartingan angle of less than about 155° in at least one orientation.

For six-membered rings, such angles can be achieved through ortho ormeta linkages to the rest of the polymer. For five-membered rings,adjacent linkages fall within this range. For eight-membered rings,linkages extending from adjacent ring atoms, from alternate ring atoms(separated by one ring atom), and from ring atoms separated by two otherring atoms fall within this range. Ring systems with more than eightring atoms may be used. For polycyclic structures, even more diverselinkage angles can be achieved.

Exemplary linking units which meet these limitations include benzenederivatives incorporated into the polymer in the 1, 2 or 1,3-positions;naphthalene derivatives incorporated into the polymer in the 1,2-, 1,3-,1,6-, 1,7-, 1,8-positions; anthracene derivatives incorporated into thepolymer in the 1,2-, 1,3-, 1,6-, 1,7-, 1,8-, and 1,9-positions; biphenylderivatives incorporated into the polymer in the 2,3-, 2,4-, 2,6-,3,3′-, 3,4-, 3,5-, 2,2′-, 2,3′-, 2,4′-, and 3,4′-positions; andcorresponding heterocycles. The position numbers are given withreference to unsubstituted carbon-based rings, but the same relativepositions of incorporation in the polymer are encompassed in substitutedrings and/or heterocycles should their distribution of substituentschange the ring numbering.

The CCP may contain at least about 0.01 mol % of the angled linker, atleast about 0.02 mol %, at least about 0.05 mol %, at least about 0.1mol %, at least about 0.2 mol %, at least about 0.5 mol %, at leastabout 1 mol %, at least about 2 mol %, at least about 5 mol %, at leastabout 10 mol %, at least about 20 mol %, or at least about 30 mol %. TheCCP may contain about 90 mol % or less, about 80 mol % or less, about 70mol % or less, about 60 mol % or less, about 50 mol % or less, or about40 mol % or less.

The CCP may be provided in isolated and/or purified form. Any suitablepurification or isolation technique may be used, alone or in combinationwith any other technique. Exemplary techniques include precipitation,crystallization, sublimation, chromatography, dialysis, extraction, etc.

The Sensor Biomolecule

A sensor biomolecule is employed that can bind to a target biomolecule.Exemplary biomolecules include polysaccharides, polynucleotides,peptides, proteins, etc. The sensor biomolecule can be conjugated to asubstrate. The sensor may also interact at least in partelectrostatically with a polycationic multichromophore, which may be aconjugated polymer. The sensor biomolecule, the target biomolecule, andthe multichromophore when bound together form a detection complex. Insome embodiments, a sensor polynucleotide is provided that iscomplementary to a target polynucleotide to be assayed, and which doesnot interact with the polycationic multichromophore in the absence oftarget to a degree that precludes the detection of target using thedescribed techniques. Desirably, in some embodiments, the sensor lacks apolyanionic backbone as found in RNA and DNA.

The sensor can be branched, multimeric or circular, but is typicallylinear, and can contain nonnatural bases. The sensor may be labeled orunlabeled with a detectable moiety.

In some embodiments, the sensor is desirably unlabelled with a moietythat absorbs energy from the multichromophore; particularly, the sensoris unlabelled with a fluorophore or quencher that absorbs energy from anexcited state of the multichromophore.

In some embodiments the sensor is labeled with a fluorophore that canabsorb energy from the multichromophore and be used in a fluorescencetransfer method for detection of polyanionic species.

The sensor may be a PNA, the molecular structures of which are wellknown. PNAs can be prepared with any desired sequence of bases. Specificsensor PNA structures can be custom-made using commercial sources orchemically synthesized.

Fluorophores

In some embodiments, a fluorophore may be employed, for example toreceive energy transferred from an excited state of the polycationicmultichromophore or visa versa, or to exchange energy with anpolynucleotide-specific dye, or in multiple energy transfer schemes.Fluorophores useful in the inventions described herein include anysubstance which can absorb energy of an appropriate wavelength and emitlight. For multiplexed assays, a plurality of different fluorophores canbe used with detectably different emission spectra. Typical fluorophoresinclude fluorescent dyes, semiconductor nanocrystals, lanthanidechelates, and green fluorescent protein.

Exemplary fluorescent dyes include fluorescein, 6-FAM, rhodamine, TexasRed, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-Chrome, phycoerythrin,PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor®430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor®568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor®660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY®FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570,BODIPY® 576/589, BODIPY® 581/591, BODIPY® 630/650, BODIPY® 650/665,BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugates thereof, andcombinations thereof. Exemplary lanthanide chelates include europiumchelates, terbium chelates and samarium chelates.

A wide variety of fluorescent semiconductor nanocrystals (“SCNCs”) areknown in the art; methods of producing and utilizing semiconductornanocrystals are described in: PCT Publ. No. WO 99/26299 published May27, 1999, inventors Bawendi et al.; U.S. Pat. No. 5,990,479 issued Nov.23, 1999 to Weiss et al.; and Bruchez et al., Science 281:2013, 1998.Semiconductor nanocrystals can be obtained with very narrow emissionbands with well-defined peak emission wavelengths, allowing for a largenumber of different SCNCs to be used as signaling chromophores in thesame assay, optionally in combination with other non-SCNC types ofsignaling chromophores.

Exemplary polynucleotide-specific dyes include acridine orange, acridinehomodimer, actinomycin D, 7-aminoactinomycin D (7-AAD),9-amino-6-chloro-2-methoxyacridine (ACMA), BOBO™-1 iodide (462/481),BOBO™-3 iodide (570/602), BO-PRO™-1 iodide (462/481), BO-PRO™-3 iodide(575/599), 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate),dihydroethidium (hydroethidine), dihydroethidium (hydroethidine),dihydroethidium (hydroethidine), ethidium bromide, ethidium diazidechloride, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2),ethidium monoazide bromide (EMA), hexidium iodide, Hoechst 33258,Hoechst 33342, Hoechst 34580, Hoechst 5769121, hydroxystilbamidine,methanesulfonate, JOJO™-1 iodide (529/545), JO-PRO™-1 iodide (530/546),LOLO™-1 iodide (565/579), LO-PRO™-1 iodide (567/580), NeuroTrace™435/455, NeuroTrace™ 500/525, NeuroTrace™ 515/535, NeuroTrace™ 530/615,NeuroTrace™ 640/660, OliGreen, PicoGreen® ssDNA, PicoGreen® dsDNA,POPO™-1 iodide (434/456), POPO™-3 iodide (534/570), PO-PRO™-1 iodide(435/455), PO-PRO™-3 iodide (539/567), propidium iodide, RiboGreen®,SlowFade®, SlowFade® Light, SYBR® Green I, SYBR® Green II, SYBR® Gold,SYBR® 101, SYBR® 102, SYBR® 103, SYBR® DX, TO-PRO®-1, TO-PRO®-3,TO-PRO®-5, TOTO®-1, TOTO®-3, YO-PRO®-1 (oxazole yellow), YO-PRO®-3,YOYO®-1, YOYO®-3, TO, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, SYTO® 9,SYTO® BC, SYTO® 40, SYTO® 41, SYTO® 42, SYTO® 43, SYTO® 44, SYTO® 45,SYTO® Blue, SYTO® 11, SYTO® 12, SYTO® 13, SYTO® 14, SYTO® 15, SYTO® 16,SYTO® 20, SYTO® 21, SYTO® 22, SYTO® 23, SYTO® 24, SYTO® 25, SYTO® Green,SYTO® 80, SYTO® 81, SYTO® 82, SYTO® 83, SYTO® 84, SYTO® 85, SYTO®Orange, SYTO® 17, SYTO® 59, SYTO® 60, SYTO® 61, SYTO® 62, SYTO® 63,SYTO® 64, SYTO® Red, netropsin, distamycin, acridine orange,3,4-benzopyrene, thiazole orange, TOMEHE, daunomycin, acridine,pentyl-TOTAB, and butyl-TOTIN. Asymmetric cyanine dyes may be used asthe polynucleotide-specific dye. Other dyes of interest include thosedescribed by Geierstanger, B. H. and Wemmer, D. E., Annu. Rev. Vioshys.Biomol. Struct. 1995, 24, 463-493, by Larson, C. J. and Verdine, G. L.,Bioorganic Chemistry: Nucleic Acids, Hecht, S. M., Ed., OxfordUniversity Press: New York, 1996; pp 324-346, and by Glumoff, T. andGoldman, A. Nucleic Acids in Chemistry and Biology, 2^(nd) ed.,Blackburn, G. M. and Gait, M. J., Eds., Oxford University Press: Oxford,1996, pp 375-441. The polynucleotide-specific dye may be anintercalating dye, and may be specific for double-strandedpolynucleotides. Other dyes and fluorophores are described atwww.probes.com (Molecular Probes, Inc.).

The term “green fluorescent protein” refers to both native Aequoreagreen fluorescent protein and mutated versions that have been identifiedas exhibiting altered fluorescence characteristics, including alteredexcitation and emission maxima, as well as excitation and emissionspectra of different shapes (Delagrave, S. et al. (1995) Bio/Technology13:151-154; Heim, R. et al. (1994) Proc. Natl. Acad. Sci. USA91:12501-12504; Heim, R. et al. (1995) Nature 373:663-664). Delgrave etal. isolated mutants of cloned Aequorea victoria GFP that hadred-shifted excitation spectra. Bio/Technology 13:151-154 (1995). Heim,R. et al. reported a mutant (Tyr66 to His) having a blue fluorescence(Proc. Natl. Acad. Sci. (1994) USA 91:12501-12504).

The Substrate

In some embodiments, the sensor is desirably located upon a substrate.The substrate can comprise a wide range of material, either biological,nonbiological, organic, inorganic, or a combination of any of these. Forexample, the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolicacid, poly(lactide coglycolide), polyanhydrides, poly(methylmethacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymericsilica, latexes, dextran polymers, epoxies, polycarbonates, orcombinations thereof. Conducting polymers and photoconductive materialscan be used.

Substrates can be planar crystalline substrates such as silica basedsubstrates (e.g. glass, quartz, or the like), or crystalline substratesused in, e.g., the semiconductor and microprocessor industries, such assilicon, gallium arsenide, indium doped GaN and the like, and includessemiconductor nanocrystals.

The substrate can take the form of a photodiode, an optoelectronicsensor such as an optoelectronic semiconductor chip or optoelectronicthin-film semiconductor, or a biochip. The location(s) of the individualsensor polynucleotide(s) on the substrate can be addressable; this canbe done in highly dense formats, and the location(s) can bemicroaddressable or nanoaddressable.

Silica aerogels can also be used as substrates, and can be prepared bymethods known in the art. Aerogel substrates may be used as freestanding substrates or as a surface coating for another substratematerial.

The substrate can take any form and typically is a plate, slide, bead,pellet, disk, particle, microparticle, nanoparticle, strand,precipitate, optionally porous gel, sheets, tube, sphere, container,capillary, pad, slice, film, chip, multiwell plate or dish, opticalfiber, etc. The substrate can be any form that is rigid or semi-rigid.The substrate may contain raised or depressed regions on which a sensorpolynucleotide or other assay component is located. The surface of thesubstrate can be etched using well known techniques to provide fordesired surface features, for example trenches, v-grooves, mesastructures, or the like.

Surfaces on the substrate can be composed of the same material as thesubstrate or can be made from a different material, and can be coupledto the substrate by chemical or physical means. Such coupled surfacesmay be composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials. The surface can be opticallytransparent and can have surface Si—OH functionalities, such as thosefound on silica surfaces.

The substrate and/or its optional surface can be chosen to provideappropriate characteristics for the synthetic and/or detection methodsused. The substrate and/or surface can be transparent to allow theexposure of the substrate by light applied from multiple directions. Thesubstrate and/or surface may be provided with reflective “minor”structures to increase the recovery of light.

The substrate and/or its surface is generally resistant to, or istreated to resist, the conditions to which it is to be exposed in use,and can be optionally treated to remove any resistant material afterexposure to such conditions.

Sensor polynucleotides can be fabricated on or attached to the substrateby any suitable method, for example the methods described in U.S. Pat.No. 5,143,854, PCT Publ. No. WO 92/10092, U.S. patent application Ser.No. 07/624,120, filed Dec. 6, 1990 (now abandoned), Fodor et al.,Science, 251: 767-777 (1991), and PCT Publ. No. WO 90/15070). Techniquesfor the synthesis of these arrays using mechanical synthesis strategiesare described in, e.g., PCT Publication No. WO 93/09668 and U.S. Pat.No. 5,384,261.

Still further techniques include bead based techniques such as thosedescribed in PCT Appl. No. PCT/US93/04145 and pin based methods such asthose described in U.S. Pat. No. 5,288,514.

Additional flow channel or spotting methods applicable to attachment ofsensor polynucleotides to the substrate are described in U.S. patentapplication Ser. No. 07/980,523, filed Nov. 20, 1992, and U.S. Pat. No.5,384,261. Reagents are delivered to the substrate by either (1) flowingwithin a channel defined on predefined regions or (2) “spotting” onpredefined regions. A protective coating such as a hydrophilic orhydrophobic coating (depending upon the nature of the solvent) can beused over portions of the substrate to be protected, sometimes incombination with materials that facilitate wetting by the reactantsolution in other regions. In this manner, the flowing solutions arefurther prevented from passing outside of their designated flow paths.

Typical dispensers include a micropipette optionally roboticallycontrolled, an ink-jet printer, a series of tubes, a manifold, an arrayof pipettes, or the like so that various reagents can be delivered tothe reaction regions sequentially or simultaneously.

The substrate or a region thereof may be encoded so that the identity ofthe sensor located in the substrate or region being queried may bedetermined. Any suitable coding scheme can be used, for example opticalcodes, RFID tags, magnetic codes, physical codes, fluorescent codes, andcombinations of codes.

Excitation and Detection

Any instrument that provides a wavelength that can excite thepolycationic multichromophore and is shorter than the emissionwavelength(s) to be detected can be used for excitation. Commerciallyavailable devices can provide suitable excitation wavelengths as well assuitable detection components.

Exemplary excitation sources include a broadband UV light source such asa deuterium lamp with an appropriate filter, the output of a white lightsource such as a xenon lamp or a deuterium lamp after passing through amonochromator to extract out the desired wavelengths, a continuous wave(cw) gas laser, a solid state diode laser, or any of the pulsed lasers.Emitted light can be detected through any suitable device or technique;many suitable approaches are known in the art. For example, afluorimeter or spectrophotometer may be used to detect whether the testsample emits light of a wavelength characteristic of the signalingchromophore upon excitation of the multichromophore. Incident lightwavelengths useful for excitation of conjugated polymers including aplurality of low bandgap repeat units can include 450 nm to 1000 nmwavelength light. Exemplary useful incident light wavelengths include,but are not limited to, wavelengths of at least about 450, 500, 550,600, 700, 800 or 900 nm, and may be less than about 1000, 900, 800, 700,600, 550 or 500 nm. Exemplary useful incident light in the region of 450nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 700 nm, and700 nm to 1000 nm. In certain embodiments the polymer forms an excitedstate upon contact with incident light having a wavelength including awavelength of about 488 nm, about 532 nm, about 594 nm and/or about 633nm. Additionally, useful incident light wavelengths can include, but arenot limited to, 488 nm, 532 nm, 594 nm and 633 nm wavelength light.

Compositions of Matter

Also provided are compositions of matter of any of the moleculesdescribed herein in any of various forms. The polymers described hereinmay be provided in purified and/or isolated form. The polymers may beprovided in crystalline form. The polymers may be provided in solution,which may be a predominantly aqueous solution, which may comprise one ormore of the additional solution components described herein, includingwithout limitation additional solvents, buffers, biomolecules,polynucleotides, fluorophores, etc. The polymers may be provided in theform of a film. The compositions of matter may be claimed by anyproperty described herein, including by proposed structure, by method ofsynthesis, by absorption and/or emission spectrum, by elementalanalysis, by NMR spectra, or by any other property or characteristic.

Methods of Use

The polymers provided herein may be employed in a variety of biologicalassays. They may be used in assays with sensor biomolecules that do notcomprise a fluorophore that can exchange energy with the conjugatedpolymer. The polymer binds at least in part electrostatically to atarget biomolecule. The target biomolecule, which may be apolynucleotide, may be labeled or unlabeled. The sensor may be bound toa substrate.

The novel polymers may also be used in biological assays in which energyis transferred between one or more of the polymer, a label on the targetbiomolecule, a label on the sensor biomolecule, and/or a fluorescent dyespecific for a polynucleotide, in all permutations. The polymer mayinteract at least in part electrostatically with the sensor and/or thetarget to form additional complexes, and an increase in energy transferwith the polymer may occur upon binding of the sensor and the target.This method may also be performed on a substrate.

Other variations of such methods are described further herein. In oneembodiment a single nucleotide polymorphism (SNP) is detected in atarget. In another embodiment expression of a gene is detected in atarget. In a further embodiment, a measured result of detecting anincrease in association of a multichromophore with a substrate can beused to diagnose a disease state of a patient. In yet another embodimentthe detection method of the invention can further include a method ofdiagnosing a disease state. In a related embodiment, the method ofdiagnosing a disease can include reviewing or analyzing data relating tothe level of association of the multichromophore with the substrate andproviding a conclusion to a patient, a health care provider or a healthcare manager, the conclusion being based on the review or analysis ofdata regarding a disease diagnosis. Reviewing or analyzing such data canbe facilitated using a computer or other digital device and a network asdescribed herein. It is envisioned that information relating to suchdata can be transmitted over the network.

Also provided is a method where a surface, which can be a sensor,changes its net charge from neutral to cationic by virtue of binding acationic multichromophore to its surface by binding of a compound to abiomolecule on the substrate.

Addition of organic solvents in some cases can result in a decrease inbackground emission by inhibiting nonionic interactions between assaycomponents, for example between the sensor and the multichromophore. Theadded solvent may be a polar organic solvent, and may be water miscible,for example an alcohol such as methanol, ethanol, propanol orisopropanol. The added solvent may be one that does not adversely affectthe ability of the sensor to hybridize to the target in the solution,for example 1-methyl-2-pyrrolidinone. The organic solvent may be addedin an amount of about 1%, about 2%, about 5%, about 10%, or more of thetotal solution, and typically is used within the range of about 0.5-10%.Other components may be incorporated into the assay solution, forexample one or more buffers suitable for maintaining a pH satisfactoryfor the biological molecules and their desired properties (e.g., abilityto hybridize).

In one variation a plurality of fluorophores, which may be directly orindirectly attached or recruited to any other of the assay componentsand/or to a substrate, can be used to exchange energy in an energytransfer scheme. In particular applications, this can provide forsignificant additional selectivity. For example, apolynucleotide-specific dye can be used as either an initial or secondsignaling fluorophore, and may be specific for double-strandedsequences. For example, energy can be transferred from an excitedcationic multichromophore to the initial signaling fluorophore, whichsubsequently transfers energy to the second signaling fluorophore, in anoverall format that is selective for the target. This cascade ofsignaling fluorophores can, in principle, be extended to use any numberof signaling fluorophores with compatible absorption and emissionprofiles. In one embodiment of this variation, an intercalating dye thatis specific for double-stranded polynucleotides is used as the secondsignaling fluorophore, and an initial signaling fluorophore that iscapable of transferring energy to the second signaling fluorophore isconjugated to the sensor polynucleotide. The intercalating dye providesthe added selective requirement that the sensor and targetpolynucleotides hybridize before it is recruited to the detectioncomplex. In the presence of target, the duplex is formed, the dye isrecruited, and excitation of the multichromophore leads to signalingfrom the second signaling fluorophore. In certain embodiments themethods of using intercalating dye(s) can include steps wherein theintercalating dye(s) is in a solution.

Any effective detection method can be used in the various methodsdescribed herein, including optical, spectroscopic, electrical,electrochemical, piezoelectrical, magnetic, Raman scattering, surfaceplasmon resonance, radiographic, colorimetric, calorimetric, etc.Preferably the sensor is or can be rendered optically detectable to ahuman and/or a detection device.

The methods described herein may be used with and incorporated into anapparatus. The methods may be used in conjunction with a commerciallyavailable device. Exemplary commercially available systems andinstruments that can be used in conjunction with an invention disclosedherein include: array systems such as the Affymetrix Genechip® system,Agilent GenePix® Microarray Scanner, Genomic Solutions GeneMachine®,Asper Biotech Genorama™ Quattroimager, and the Bio-Rad VersArray®ChipReader; and real time PCR systems such as the Applied Biosystems7900HT Fast Real-Time PCR System, ABI PRISM® 7000 Sequence DetectionSystem, Applied Biosystems 7500 Real-Time PCR System, Applied Biosystems7300 Real-Time PCR System, Applied Biosystems PRISM® 7700, Bio-Rad MyiQSingle-Color Real-Time PCR Detection System, and the Bio-Rad iCycler iQReal-Time PCR Detection System.

Articles of Manufacture

The CPs can be incorporated into any of various articles of manufactureincluding optoelectronic or electronic devices, biosensors, diodes,including photodiodes and light-emitting diodes (“LEDs”), optoelectronicsemiconductor chips, semiconductor thin-films, and chips, and can beused in array or microarray form. The polymer can be incorporated into apolymeric photoswitch. The polymer can be incorporated into an opticalinterconnect or a transducer to convert a light signal to an electricalimpulse. The CPs can serve as liquid crystal materials. The CPs may beused as electrodes in electrochemical cells, as conductive layers inelectrochromic displays, as field effective transistors, and as Schottkydiodes.

The CPs can be used as lasing materials. Optically pumped laser emissionhas been reported from MEH-PPV in dilute solution in an appropriatesolvent, in direct analogy with conventional dye lasers [D. Moses, Appl.Phys. Lett. 60, 3215 (1992); U.S. Pat. No. 5,237,582]. Semiconductingpolymers in the form of neat undiluted films have been demonstrated asactive luminescent materials in solid state lasers [F. Hide, M. A.Diaz-Garcia, B. J. Schwartz, M. R. Andersson, Q. Pei, and A. J. Heeger,Science 273, 1833 (1996); N. Tessler, G. J. Denton, and R. H. Friend,Nature 382, 695 (1996)]. The use of semiconducting polymers as materialsfor solid state lasers is disclosed in U.S. Pat. No. 5,881,083 issuedMar. 9, 1999 to Diaz-Garcia et al. and titled “Conjugated Polymers asMaterials for Solid State Lasers.” In semiconducting polymers, theemission is at longer wavelengths than the onset of significantabsorption (the Stokes shift) resulting from inter- and intramolecularenergy transfer. Thus there is minimal self-absorption of the emittedradiation [F. Hide et al., Science 273, 1833 (1996)], so self-absorptiondoes not make the materials lossy. Moreover, since the absorption andemission are spectrally separated, pumping the excited state via the πto π* transition does not stimulate emission, and an inverted populationcan be obtained at relatively low pump power.

Light-emitting diodes can be fabricated incorporating one or more layersof CPs, which may serve as conductive layers. Light can be emitted invarious ways, e.g., by using one or more transparent or semitransparentelectrodes, thereby allowing generated light to exit from the device.

The mechanism of operation of a polymer LED requires that carrierinjection be optimized and balanced by matching the electrodes to theelectronic structure of the semiconducting polymer. For optimuminjection, the work function of the anode should lie at approximatelythe top of the valence band, E_(v), (the π-band or highest occupiedmolecular orbital, HOMO) and the work function of the cathode should lieat approximately the bottom of the conduction band, E_(c), (the π-bandor lowest unoccupied molecular orbital, LUMO).

LED embodiments include hole-injecting and electron-injectingelectrodes. A conductive layer made of a high work function material(above 4.5 eV) may be used as the hole-injecting electrode. Exemplaryhigh work function materials include electronegative metals such as goldor silver, and metal-metal oxide mixtures such as indium-tin oxide. Anelectron-injecting electrode can be fabricated from a low work functionmetal or alloy, typically having a work function below 4.3. Exemplarylow work function materials include indium, calcium, barium andmagnesium. The electrodes can be applied by any suitable method; anumber of methods are known to the art (e.g. evaporated, sputtered, orelectron-beam evaporation).

In some embodiments, polymer light-emitting diodes have been fabricatedusing a semiconducting polymer cast from solution in an organic solventas an emissive layer and a water-soluble (or methanol-soluble)conjugated copolymer as an electron-transport layer (ETL) in the deviceconfiguration: ITO(indium tin oxide)/PEDOT(poly(3,4-ethylenedioxythiophene)/emissive polymer/ETL/Ba/Al. The inventors havesuccessfully fabricated multi-layer PLEDs using a semiconducting polymer(red, green or blue emitting), cast from solution in an organic solvent,as the emissive layer and a water-soluble (or methanol-soluble) cationicconjugated copolymer as electron-transport layer. The resultsdemonstrate that devices with the ETL have significantly lower turn-onvoltage, higher brightness and improved luminous efficiency.

Although the examples demonstrate the use of an electron-transport layerformed from the soluble conductive polymer, any form of conducting layercan be used. Thus, judicious choice of monomers as described herein canresult in polymers with hole-injecting and/or transporting properties,as well as polymers with electron-injecting and/or transportingproperties. The device geometry and deposition order can be selectedbased on the type of conductive polymer being used. More than one typeof conductive polymer can be used in the same multilayer device. Amultilayer device may include more than one layer of electron-injectingconjugated polymers, more than one layer of hole-injecting conjugatedpolymers, or at least one layer of a hole-injecting polymer and at leastone layer of an electron-injecting conjugated polymer.

In PLEDs, the device efficiency is reduced by cathode quenching sincethe recombination zone is typically located near the cathode.^([20]) Theaddition of an ETL moves the recombination zone away from the cathodeand thereby eliminates cathode quenching. In addition, the ETL can serveto block the diffusion of metal atoms, such as barium and calcium, andthereby prevents the generation of quenching centers^([20]) during thecathode deposition process.

In some embodiments, the principal criteria when a soluble conjugatedpolymer is used as an electron transport layer (ETL) in polymerlight-emitting diodes (PLEDs) are the following: (1) The lowestunoccupied molecular orbital (LUMO) of the ETL must be at an energyclose to, or even within the π*-band of the emissive semiconductingpolymer (so electrons can be injected); and (2) The solvent used forcasting the electron injection material must not dissolve the underlyingemissive polymer.

Similarly, the principal criteria for a polymer based hole transportlayer (HTL) for use in polymer light-emitting diodes (PLEDs) is that thehighest occupied molecular orbital (HOMO) of the HTL must be at anenergy close to, or even within the valence band of the emissivesemiconducting polymer.

Solubility considerations can dictate the deposition order of theparticular CPs ans solvents used to produce a desired deviceconfiguration. Any number of layers of CPs with different solubilitiesmay be deposited via solution processing by employing these techniques.

The PLEDs comprising CPs described herein can be incorporated in anyavailable display device, including a full color LED display, a cellphone display, a PDA (personal digital assistant), portable combinationdevices performing multiple functions (phone/PDA/camera/etc.), a flatpanel display including a television or a monitor, a computer monitor, alaptop computer, a computer notepad, and an integrated computer-monitorsystems. The PLEDs may be incorporated in active or passive matrices.

FIG. 9 is a block diagram showing a representative example logic devicethrough which reviewing or analyzing data relating to the presentinvention can be achieved. Such data can be in relation to a disease,disorder or condition in a subject. FIG. 9 shows a computer system (ordigital device) 100 that may be understood as a logical apparatus thatcan read instructions from media 111 and/or network port 105, which canoptionally be connected to server 109 having fixed media 112. The systemshown in FIG. 9 includes CPU 101, disk drives 103, optional inputdevices such as keyboard 115 and/or mouse 116 and optional monitor 107.Data communication can be achieved through the indicated communicationmedium to a server 109 at a local or a remote location. Thecommunication medium can include any means of transmitting and/orreceiving data. For example, the communication medium can be a networkconnection, a wireless connection or an internet connection. It isenvisioned that data relating to the present invention can betransmitted over such networks or connections.

In one embodiment, a computer-readable medium includes a medium suitablefor transmission of a result of an analysis of a biological sample. Themedium can include a result regarding a disease condition or state of asubject, wherein such a result is derived using the methods describedherein.

Kits

Kits comprising reagents useful for performing described methods arealso provided.

In some embodiments, a kit comprises a polycationic multichromophore asdescribed herein and one or more substrate-bound unlabeledsingle-stranded sensor polynucleotides that are complementary tocorresponding target polynucleotide(s) of interest. In the presence ofthe target polynucleotide in the sample, the sensor is brought intoproximity to the multichromophore upon hybridization to the target,which associates electrostatically with the polycationicmultichromophore. Association of the multichromophore with the sensorpermits detection and/or quantitation of the target in the sample.

In some embodiments, a kit comprises a polycationic conjugated polymerhaving an absorption of greater than about 450 nm that can increasesignal amplification and one or more sensor biomolecules that can bindto target biomolecule(s) of interest. In these embodiments, associationof the multichromophore with the sensor can be directly detected orindirectly detected through energy transfer to or from another species,for example a fluorescent label conjugated to the target and/or asubstrate, or to or from an intercalating dye that can intercalate withthe sensor-target bound complex.

In some embodiments, a kit comprises an aqueous solution of apolycationic conjugated polymer having an absorption of greater thanabout 450 nm and one or more sensor biomolecules that can bind to targetbiomolecule(s) of interest. In these embodiments, association of themultichromophore with the sensor can be directly detected or indirectlydetected through energy transfer to another species, for example afluorescent label conjugated to the target and/or a sensor and/or asubstrate to which it may be attached, or to or from an intercalatingdye that can intercalate with the sensor-target bound complex.

The kit may optionally contain one or more of the following: one or morelabels that can be incorporated into a target; one or more intercalatingdyes; one or more sensor biomolecules, one or more substrates which maycontain an array, etc.

The components of a kit can be retained by a housing. Instructions forusing the kit to perform a described method can be provided with thehousing, and can be provided in any fixed medium. The instructions maybe located inside the housing or outside the housing, and may be printedon the interior or exterior of any surface forming the housing whichrenders the instructions legible. A kit may be in multiplex form fordetection of one or more different target polynucleotides or otherbiomolecules.

As described herein and shown in FIGS. 10A and 10B, in certainembodiments a kit (200, 300) for assaying a sample is provided. The kit(200, 300) can include a container (208, 308) for containing variouscomponents. As shown in FIG. 10B, in one embodiment a kit 300 forassaying a sample for a target includes a container 308, a polymer 304and a sensor 302. The target can be a biomolecule including but notlimited to a polynucleotide. Polymer 304 can include but is not limitedto a polycationic conjugated polymer or a dendrimer. In one embodimentpolymer 304 is a polycationic multichromophore. Sensor 302 can be abiomolecule including but not limited to a polynucleotide complementaryto the target. As shown in FIG. 10A, in one embodiment, kit 200 includesa container 208, a polymer 204 and a sensor 202 conjugated to asubstrate. Polymer 204 can include but is not limited to a polycationicconjugated polymer or a dendrimer. In one embodiment, polymer 204 is apolycationic multichromophore. Sensor 202 can be a biomolecule includingbut not limited to a polynucleotide complementary to the target. Asshown in FIGS. 10A and 10B, the kit (200, 300) can optionally includeinstructions (206, 306) for using the kit (200, 300). Other embodimentsof the kit (200, 300) are envisioned wherein the components includevarious additional features described herein.

EXAMPLES

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete description of how to make and use thepresent invention, and are not intended to limit the scope of what isregarded as the invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessotherwise indicated, parts are parts by weight, temperature is degreecentigrade and pressure is at or near atmospheric, and all materials arecommercially available.

Example 1 Synthesis of2,7-bis[9,9′-bis(6″-bromohexyl)-fluorenyl]-4,4,5,5-tetramethyl-[1.3.2]dioxaborolane

A 50 mL round bottomed flask was charged with9,9′-bis(bromohexyl)-2,7-dibromofluorene (0.65 g, 1 mmol) in 20 mL ofdry THF and cooled to −78° C. with a dry ice/acetone bath. At −78° C., 3eq. of t-BuLi in pentane (1.8 mL, 1.7 M) was added drop by drop and itwas followed immediately by adding2-isopropoxy-4,4,5,5-tetramethyl-[1.3.2]-dioxabororane (1.3 mL, 6 mol)in one shot. The resulting solution was slowly warmed to roomtemperature and stirred overnight. It was quenched by water, and thesolution was concentrated by rotary evaporation and extracted withchloroform. The organic phase was separated and dried over magnesiumsulfate. After evaporation, the reside was purified with silica gelcolumn chromatography (ethyl acetate/hexane 1:20) to yield 0.15 g (20%)of the product as white crystals. ¹H-NMR (400 MHz, CDCl₃): δ 7.83-7.72(m, 6H), 3.27-3.24 (t, J=6.8 Hz, 4H), 2.03-1.99 (q, J=4.0 Hz, 4H),1.64-1.57 (q, J=7.2 Hz, 4H), 1.39 (s, 24H), 1.17-1.13 (q, 4H), 1.06-1.02(q, 4H), 0.55 (m, 4H). This monomer is within the scope of theinvention, and may be claimed by its proposed structure describedherein, by its method of production,

Example 2 Synthesis of PFBT

The synthesis ofpoly[9,9′-bis(6″-N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole)dibromide] (“PFBT”) is shown in Scheme 1. Suzuki copolymerization of2,7-bis[9,9′-bis(6″-bromohexyl)-fluorenyl]-4,4,5,5-tetramethyl-[1.3.2]dioxaborolaneand 4,7-dibromo-2,1,3-benzothiadiazole givespoly[9,9′-bis(6″-bromohexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole)]in 65% yield.¹⁷ The resulting polymer was purified by three timesprecipitation from chloroform to methanol. Elemental analysis and ¹H and¹³C NMR spectroscopies are consistent with the structure in Scheme 1.Gel permeation chromatography shows a number average molecular weight of12,000 and a PDI of 1.7, relative to polystyrene standards. In a secondstep, nucleophilic displacement using trimethylamine generates thecationic charges on the polymer pendant groups and produces PFBT, whichis soluble in aqueous media.

Poly[9,9′-bis(6′-bromohexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole)](PFBT precursor) was synthesized as follows.2,7-Bis[9,9′-bis(6″-bromohexyl)-fluorenyl]-4,4,5,5-tetramethyl-[1.3.2]dioxaborolane(186 mg, 0.25 mmol) and 4,7-dibromo-2,1,3-benzothiadiazole (73.5 mg,0.25 mmol), Pd(PPh₃)₄ (5 mg) and potassium carbonate (830 mg, 6 mmol)were placed in a 25 mL round bottom flask. A mixture of water (3 mL) andtoluene (6 mL) was added to the flask and the reaction vessel wasdegassed. The resulting mixture was heated at 85° C. for 20 h, and thenadded to acetone. The polymer was filtered and washed with methanol andacetone, and then dried under vacuum for 24 h to afford PFBT (105 mg,65%) as a bright yellow powder. ¹H NMR (200 MHz, CDCl₃): δ 8.07-7.91 (m,6H), 7.87-7.72 (m, 2H) 3.36-3.29 (t, 6H, J=6.6 Hz), 2.19 (m, 4H), 1.7(m, 4H), 1.3-1.2 (m, 8H), 0.9 (m, 4H). ¹³C NMR (50 MHz, CDCl₃): δ 154.5,151.6, 141.2, 136.8, 133.8, 128.5, 124.3, 120.6, 55.6, 40.4, 34.3, 32.9,29.3, 27.9, 23.9. GPC (THF, polystyrene standard), M_(w): 20500 g/mol;M_(n): 12,000 g/mol; PDI: 1.7. Elemental Analysis Calculated: C, 59.43;H, 5.47; N. 4.47. Found: C, 60.13; H, 5.29; N, 3.35.

Poly[9,9′-bis(6″-N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole)dibromide] (PFBT) was synthesized as follows. Condensed trimethylamine(2 mL) was added dropwise to a solution of the neutral precursor polymer(70 mg) in THF (10 mL) at −78° C. The mixture was then allowed to warmup to room temperature. The precipitate was re-dissolved by addition ofwater (10 mL). After the mixture was cooled down to −78° C., moretrimethylamine (2 mL) was added and the mixture was stirred for 24 h atroom temperature. After removing most of the solvent, acetone was addedto precipitate PFBT (72 mg, 89%) as a light brown powder. ¹H NMR (200MHz, CD₃OD): δ 8.30-7.80 (m, 8H), 3.3-3.2 (t, 4H), 3.1 (s, 18H), 2.3(br, 4H), 1.6 (br, 4H), 1.3 (br, 8H), 0.9 (br, 4H). ¹³C NMR (50 MHz,CD₃OD): δ 155.7, 152.7, 142.7, 138.2, 134.6, 129.9, 125.3, 121.5, 67.8,56.9, 52.5, 41.4, 30.5, 27.1, 25.2, 23.9.

Example 3 Absorption and Emission Spectra of PFBT

FIG. 2 shows the absorption and emission spectra of PFBT in 25 mMphosphate buffer (pH=7.4) and as a film obtained by casting the polymerfrom a methanol solution (0.005 M). The absorption spectra of PFBT, bothin solution and in the solid, display two well-separated bands. Theabsorption band centered at 330 nm is attributed to the fluorenesegments in the polymer, while the absorption at 455 nm corresponds tothe contribution from the benzothiadiazole units.¹⁸ The PL spectra forPFBT do not show vibronic structure and display maxima at approximately590 nm.¹⁹ PFBT has a quantum yield of 10±2% in water (using fluoresceinat pH=11 as a standard). The solid-state quantum yield of PFBT wasmeasured to be 4±1% in the solid-state by using an integrating sphere.FIG. 2 also contains the absorption and emission of a Cy-5 labeled PNA(PNA_(I)-Cy5=5′-Cy5-CAGTCCAGTGATACG-3′). Examination of the 300 to 700nm range, shows that only PFBT absorbs at 488 and that there isexcellent overlap between the emission of PFBT and the absorption ofCy-5, a necessary condition for FRET from PFBT to Cy-5.

Example 4 Use of PFBT For Polynucleotide Detection

The use of PFBT (3.0×10⁻⁷ M in repeat units, RUs) and PNA_(I)-Cy5([PNA_(I)-Cy5]=1×10⁻⁹ M) in the assay shown in FIG. 1 was tested using acomplementary ssDNA (ssDNA_(I)c=5′-CGTATCACTGGACTG-3′) and a noncomplementary ssDNA (ssDNA_(I)n=5′-CAGTCTATCGTCAGT-3′). The experimentalconditions chosen include use of a 25 mM phosphate buffer (pH=7.4) with5% 1-methyl-2-pyrrolidinone (NMP). The small quantity of organic solventreduces hydrophobic interactions between PNA and PFBT. As shown in FIG.3, the addition of PFBT to ss-DNA_(I)c/PNA_(I)-Cy5 followed byexcitation at 460 nm results in intense red emission from the Cy5. Thereis no energy transfer for the solution containing ssDNA_(I)n/PNA_(I)-Cy5under these experimental conditions.

Example 5 Use of PFBT for Detection on a Substrate

To demonstrate that the signal amplification afforded by the lightharvesting properties of PFBT could be incorporated into platformssuitable for microarray technologies, we designed a simplified teststructure with PNA probes attached to glass or silica surfaces, as shownin FIG. 4. The test surface was prepared by a sequence of steps thatbegins with treatment using 2-aminopropyltrimethoxysilane and subsequentactivation with 1,4-phenylenediisothiocyanate (PDITC). Amine terminatedPNA (PNA_(II)═NH₂—O—O-TCCACGGCATCTCA), where O corresponds to a C₆H₁₁NO₃linker fragment, was immobilized on the activated surface by takingadvantage of well established isothiocyanate/amine coupling protocols.²⁰

To estimate the amount of ssDNA that the PNA-containing surfaces cancapture, they were treated with a dye-labeled ssDNA(ssDNA_(II)c-Cy5=5′-Cy5-TGAGATGCCGTGGA, [ssDNA_(II)c-Cy5]=3×10⁻⁷ M)solution for 30 minutes at room temperature, followed by washing steps(see Supplementary Information). The resulting quantity of ssDNAc wasestimated by measuring the Cy5 fluorescence with a fluoroimager.Comparison of the resulting Cy5 emission intensities against theintensities from a known number of chromophores provided an estimate of10¹² strands of hybridized ssDNA_(II)c-Cy5 per cm². Cy5 emission was notdetected when the surface was treated with the non-complementaryssDNA_(II)n-Cy5 (ssDNA_(II)n-Cy5=5′-Cy5-ATCTTGACTGTGTGGGTGCT-3′).

FIG. 5 illustrates the anticipated function of the PNA/CCP assay in thesolid-state. Treatment of the PNA (a) containing surface withcomplementary ssDNA (b, left, shown as polyanionic) increases thenegative surface charge. Addition of the CCP results in binding to thesurface. Excitation of the polymer results in FRET to the reporter dye.The purpose of the diagram is to show the molecular components in thesystem and the main recognition/electrostatic events in the sensoroperation and not to imply molecular orientations relative to thesurface. When non-complementary DNA is used (not shown), the reporterdye is not incorporated onto the surface.

FIG. 6 shows the Cy5 emission measured with a standard fluorometer frompost-hybridization PNA/DNA substrates after addition of PFBT (1 μL,[PFBT]=4×10⁻⁶ M). Cy5 emission is clearly detected from thessDNA_(II)c-Cy5/PNA_(II)/PFBT surface (a), while none is observed fromthe ssDNA_(II)n-Cy5/PNA_(II)/PFBT substrate (b). That Cy5 emission isobserved in (a) but not (b) reflects the PNA/DNA selectivity. Theadditional sensitivity afforded by the addition of PFBT can beestablished by excitation of the ssDNA_(II)c-Cy5/PNA_(II)/PFBT surfaceat 470 nm (PFBT absorption) and at 645 nm (Cy5 absorption maximum). Overan order of magnitude amplification of the dye emission is observed.These results confirm the operation of the sensor as illustrated in FIG.1.

Example 6 Target Polynucleotide Detection Using a Second PolycationicMultichromophore

Because PFBT can be excited at 488 nm and its solid-state emission canbe detected with a commercial fluoroimager, it enables a solid-stateassay that does not require using labeled sensor polynucleotides fordetection of a desired target. The overall process is illustrated inFIG. 7. In one embodiment, hybridization of ssDNA to the PNA surface(FIG. 7 a) results in a negatively charged surface (FIG. 7 b). Becauseof electrostatic attraction, the addition of the CCP, followed bywashing, should result in preferential adsorption on those sites thatcontain the complementary ssDNA (FIG. 7 c). After workup, polymeremission indicates that the ssDNA is complementary to the PNA sequence.The overall selectivity of FIG. 7 relies on the successful removal ofCCP from non-hybridized PNA surfaces.

FIG. 8 shows the integrated PFBT emission from 1 mm² PNA_(II) surfacestreated according to FIG. 7. The polymer was deposited by addition of0.5 μL of a 5×10⁻⁷ M solution and was allowed to stand in ahybridization chamber for thirty minutes at room temperature. Washingwas accomplished by using SSC (1×)+0.1% SDS solution, followed by SSC(0.1×). When placed inside a fluoroimager one can observe that the PFBTemission from the PNA_(II)/ssDNA_(II)c surfaces is approximately fivetimes more intense than the emission from the PNA_(II)/ssDNA_(II)nareas. Exposing the surfaces to more concentrated PFBT solutions resultsin more intense emission signals, but poorer selectivities. The data inFIG. 8 demonstrate that it is possible to detect the presence of 10¹⁰ssDNA strands that are complementary to the surface PNA sequence,without the need to label the target species.

Example 7 Use of PFBT for Detection of Polynucleotides on DNA SensorArrays

A far-red labeled target polynucleotide was probed using DNA sensorsbound to a substrate. When the slide is treated with PFBT polymer thosesensor spots which specifically bind a labeled target polynucleotideshow increased signal from the target label when the PFBT is excited.Spots without bound target do not show appreciable signal in thewavelength range of the target label allowing one to easilydifferentiate sensor and target binding events.

Probe Immobilization: Functionalized isothiocyanate slides (SAL-1, AsperBiotech) were used for binding amine terminal sensor molecules. Theamino-functionalized DNA (IDT) probe was dissolved in H₂O to aconcentration of 1×10⁻⁴ M and then diluted to a final concentration of5×10⁻⁵ M in 50 mM Na₂CO₃/NaHCO₃ buffer (pH 9.0). Spotting wasaccomplished using 1 μl aliquots with a standard micropipette. Bindingof DNA to the surface was performed at 37° C. over a period of 3 hrsinside a humid Corning hybridization chamber containing saturated NaClsolution. After 3 hrs of incubation, the slides were rinsed with DIwater followed by rinsing with methanol. The surface was thendeactivated in a solution made of dimethylformamide (50 ml) withaminoethanol (0.5 ml) and diisopropylethylamine (1 ml) over a period of1 hr under shaking. The slides were subsequently washed withdimethylformamide, acetone and water before drying with a flow ofnitrogen.

Target hybridization: Target hybridization was performed in the Corninghybridization chamber by using Alexa 750 labeled complimentary DNA at aconcentration of 2×10⁻⁶M in 3×SSC at 42° C. over a period of 16 hrs indarkness. After the 16 hrs incubation, the slide was sequentially washedwith 1×SSC/0.1% SDS for 10 min at 42° C., 1×SSC/0.1% SDS for 5 min at42° C., 0.1×SSC/0.1% SDS for 5 min at room temperature, and 0.1×SSC/0.1%SDS for 5 min at room temperature. The slide was then rinsed with DIwater and transferred to a clean tube containing a solution of PFBTpolymer.

Polymer Binding: The slide was put into a solution of polymer (about1×10⁻⁶ M in repeat units) and incubated at room temperature undershaking for 20 min. After incubation in the polymer solution, the slidewas washed with 1×SSC for 5 min at room temperature, followed by a waterrinse and a methanol wash for 10 min at room temperature. After themethanol wash, the slide was dried under a flow of nitrogen.

Array Scanning: Fluorescence scanning was done on a 4-laser ProScanArray(Perkin Elmer) using a setting of 80% for the laser power and a PMT gainsetting of 40%. The excitation wavelength used was 488 nm for thepolymer and 633 nm for Alexa 750. Emission of the polymer was measuredat 578 nm and the emission of the Alexa 770 was measusued at 780 nm.Data analysis was done with the ScanArray Express Microarray AnalysisSystem. Signal obtained was the mean signal intensity of each spot underspecific excitation and emission settings. FRET scans were performedusing 488 nm excitation and 780 nm emission settings. Sensor spots withbound Alexa 750 labeled target DNA provided a high FRET signal and alowered polymer signal allowing clear distinction of target bound spots.The FRET signals generated with PFBT polymer excitation provided Alexa750 signals over 50% greater than the directly excited Alexa 750.

Example 8 Use of PFBT Multichromophore on Substrate Bound MultiplexDetection Probes

To demonstrate the integration of the PFBT multichromophore on asubstrate comprised of multiple human genome sensor polynucleotides, amicroarray slide was prepared in the following manner and tested onlabeled target cDNA:

Target hybridization: Target hybridization was performed on a MWG HumanStarter Array slide with a LifterSlip (22×22 mm) in a Corninghybridization chamber using Alexa 647 labeled human cDNA. The targetsample was prepared by diluting 10 ul of the Alexa 647 labeled cDNA with20 ul of 8×SSPE/1.5% Tween 20 and heated up to 95° C. for 5 min. After abrief spin, the target cDNA was immediately loaded onto the slide usinga LifterSlip. The hybridization chamber was quickly assembled andincubated at 42° C. over a period of 16 hrs in darkness. After the 16hrs incubation, the LifterSlip was first rinsed with 5×SSPE/0.1% Tween20 at 42° C. and the slide was sequentially washed with 5×SSPE/0.1%Tween 20 for 10 min at 42° C., 5×SSPE/0.1% Tween 20 for 10 min at 42°C., 1×SSPE/0.1% Tween 20 for 10 min at room temperature and three 1 minwashes with 0.1×SSPE/0.1% Tween 20 at room temperature. The slide wasthen rinsed with DI water and incubated in 10% PEG for 2 hrs. After thePEG incubation, the slide was quickly rinsed with water and transferredto a clean tube containing the PFBT polymer solution.

Polymer Binding: The slide was placed into a solution of polymer (about1×10⁻⁶ M in repeat units) and incubated at room temperature undershaking for 20 min. After incubation in the polymer solution, the slidewas washed with 1×SSC for 15 min at 37° C., followed by a 1×SSC rinse, awater rinse and a methanol wash each for 10 min at room temperature.After the methanol wash, the slide was dried under a flow of nitrogen.

Array Scanning: Fluorescence scanning was done on a 4-laser ProScanArray(Perkin Elmer) using a setting of 80% for the laser power. Excitationwavelength used was 488 nm for the polymer and 633 nm for Alexa 647.Emission of the polymer was measured at 578 nm with a PMT gain of 35%and emission of the Alexa 647 was measured at 670 nm with a PMT gain of50%. Data analysis was done with the ScanArray Express MicroarrayAnalysis System. Signal obtained was the mean signal intensity of eachspot under specific excitation and emission settings. FRET scans wereperformed using 488 nm excitation and 670 nm emission settings with aPMT gain of 35%. Sensor spots binding higher amounts of target cDNAlabeled with Alexa647 provided a higher FRET signal and a lower polymersignal allowing clear distinction of target bound spots.

Although the invention has been described in some detail with referenceto the preferred embodiments, those of skill in the art will realize, inlight of the teachings herein, that certain changes and modificationscan be made without departing from the spirit and scope of theinvention. Accordingly, the invention is limited only by the claims.

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1. An assay method comprising: providing a sample that is suspected ofcontaining a target polynucleotide; providing a polycationicmultichromophore that comprises a conjugated polymer or dendrimer,wherein said multichromophore has a detectable excitation emission;providing a sensor that is a polynucleotide that is complementary to thetarget polynucleotide, wherein said sensor is conjugated to a substrateand is not conjugated to a fluorescently detectable label that canreceive energy from an excited state of the multichromophore; contactingthe substrate with the sample; contacting the substrate with themultichromophore under conditions wherein the multichromophore can bindto the sensor and/or target polynucleotide, if present; detectingwhether target has bound to the sensor.
 2. The method of claim 1,wherein detecting whether target has bound to the sensor comprisesapplying a light source that can excite the multichromophore anddetecting whether light is emitted in the vicinity of the substrate. 3.The method of claim 1, wherein detecting whether target has bound to thesensor comprises applying a light source that can excite themultichromophore and detecting whether energy has been transferred fromthe multichromophore.
 4. The method of claim 3, wherein the methodcomprises detecting whether energy has been transferred from themultichromophore to a fluorophore.
 5. The method of claim 1, wherein thepolycationic multichromophore has a detectable excitation emission whenexcited with light comprising a wavelength in the region of about 450 nmto about 1000 nm.
 6. The method of claim 2, the method comprisingdetecting whether the multichromophore emits light in the vicinity ofthe substrate.
 7. The method of claim 1, wherein the multichromophorecomprises a conjugated polymer.
 9. The method of claim 1, wherein lightemitted from the multichromophore above a threshold level indicates thatthe target polynucleotide is present in the sample.
 10. The method ofclaim 1, wherein the amount of light emitted from the multichromophoreis quantitated and used to determine the amount of the targetpolynucleotide in the sample.
 11. A kit for assaying a sample for atarget polynucleotide comprising: a conjugated polymer and that caninteract with a target polynucleotide; a substrate comprising a sensorpolynucleotide that is complementary to the target polynucleotide,wherein said sensor polynucleotide is not conjugated to a fluorescentlydetectable label that can receive energy from an excited state of themultichromophore.
 12. The kit of claim 11, further comprising aplurality of different sensor polynucleotides complementary to acorresponding plurality of different target polynucleotides conjugatedto the substrate.
 13. A detection method comprising: providing a samplethat is suspected of containing a target biomolecule; providing achromophore that comprises a conjugated polymer, wherein saidchromophore has a detectable excitation emission; providing a sensorbiomolecule capable of recognition pairing with the target biomolecule,wherein the sensor is conjugated to a substrate and is not conjugated toa fluorescently detectable label that can receive energy from an excitedstate of the chromophore; contacting the substrate with the sample underconditions in which the sensor can pair with the target, if present;contacting the substrate with the multichromophore under conditionswherein the multichromophore can bind to the sensor and/or targetpolynucleotide, if present; detecting whether target has bound to thesensor.
 14. The method of claim 13, wherein the substrate is furthercontacted with an intercalating dye in a solution under conditions inwhich the dye can intercalate with the sensor and the target when pairedand wherein the target is conjugated to a fluorescently detectable labelthat can receive energy from an excited state of the chromophore. 15.The method of claim 13, wherein the sensor is selected from the groupconsisting of a peptide and a polynucleotide.
 16. The method of claim13, wherein the chromophore has a detectable excitation emission whenexcited with light having a wavelength in the region of about 450 nm toabout 1000 nm.